Proof of Concept

The RUM-UPRM has taken upon the job of theoretically implementing a fluorometric assay in a 96-well plate reader, a high-performance liquid chromatography (HPLC), and the use of an UV spectrophotometer to confirm the functionality of our genetic circuit. For us to analyze the performance of each assembly containing our Device 1 and Device 2 in their respective plasmids, we opted to use a modification of last cycle’s protocol related to the fluorometric assay. The fluorometric assay in a 96 well plate reader will estimate the optimal absorbance that emits the red fluorescence protein (RFP) from Device 1 and the reporter gene amilGFP from Device 2. The colonies producing the correct absorbance of the fluorescence wavelength will be inoculated for the HPLC procedure.

As designed; the Device 2 is responsible for degrading the RDX contaminant. Therefore, the HPLC procedure will be responsible for quantifying the RDX present in the sample over specific time intervals. This will confirm that the xplB and xplA gene complex is aerobically degrading by double denitrification; lowering the concentration of RDX in the sample. For future implementation, a UV spectrophotometer is going to be used to measure the performance of our last device of the genetic circuit; the Killswitch. The UV spectrophotometer will be in charge of calculating the turbidity or optical density of a culture broth through the amount of light that is being transmitted in the sample. This will help us estimate the cell biomass and timelapse of cellular growth of the experimental sample containing our Killswitch.

Our team will use EC100 cells grown in a RDX infused LB broth (Luria bertani broth) with Chloramphenicol as an antibiotic to prepare the liquid medium experimental sample for the fluorometric assay for Device 1 and Device 2. Based on the methods used on Pseudomonas, a concentration of 0.2-0.5mM of RDX will be applied as a nitrogen source in the medium (Lee et al., 2013). Additionally, a LB broth liquid medium with Chloramphenicol will be inoculated with EC100 cells for our control sample. The medium will be placed at 30ºC on a rotary shaker at 150rpm (Lee et al., 2013). Later on, we will quantify this data by composing a spectrum graph where it compares wavelength vs transmittance. The spectrum graph will demonstrate the accurate absorbed wavelength according to our reporter genes.

Device 1 has a red fluorescence protein (RFP) as a biological marker for the confirmation of DNA transcription. The RFP (BBa_K081014) is a highly engineered mutant of a monomeric red fluorescent protein from the coral Discosoma striata (Part:BBa E1010 - Parts.igem.org, 2013). Therefore, the RFP will transmit a red fluorescence that counts with an excitation peak of 584nm and an emission peak of 607nm which will be used as a criterion for the absorbance variable when selecting the colonies. The selected colonies should emit the equitable emission and excitation wavelengths of the RFP in order to be inculcated for the HPLC procedure. On the other hand, the Device 2 includes the reporter gene amilGFP (BBa_K592010) that will display a yellow chromaticity that could be observed when performing a fluorometric assay. The natural yellow fluorescence of the amilGFP is conveyed because it is a chromoprotein from the coral Acropora millepora. Hence, the amilGFP will transmit an absorption peak of 502nm and an emission peak of 512nm.

In the same manner we plan on using the fluorometric assay as a method to measure the absorbance variable when selecting the colonies on Device 1, we expect to achieve the same with Device 2. In synthesis, the fluorometric assay will be performed in order to qualitatively observe which colonies are emitting the correct wavelength according to the values of emission, absorption, and excitation of the biological markers of their respective devices. This will prove that the RDX in the medium is inducing the transcription of the genetic circuits placed inside the EC100 cells, consequently activating the emittance of fluorescent light of the biological markers.

Figure 1. Fluorometry Assay in a 96 Well Plate Reader Procedure.

The colonies selected after the fluorometric assay will be inoculated in order to execute the HPLC procedure. The HPLC will be done to quantify the concentration of RDX in the sample through time for the confirmation of the degradation complex of our Device 2. The concentration of RDX will be quantified in 0.5 hours intervals for 48h. Based on Panchal et al., 2017 methodology for HPLC analysis, our team will use a C8 column (4.6 x 250 mm, 5 µm) with detection at 208 nm. Isocratic separations are performed using a 0.05% TFA:acetonitrile (60:40 v/v) mobile phase with a flow rate of 1 ml/min and the injection volume is 5 µl with a temperature set at 25 °C (Panchal et al., 2017). We must mention that for our control sample we will not be applying the inductor RDX in the medium for the injection into the HPLC sample. For the analysis of the results obtained from the HPLC procedure we will compose a validation curve of concentration vs time for each colony tested.

Figure 2. High Performance Liquid Chromatography (HPLC) Procedure.

Our Device 3; that is in charge of bacterial lysis, will be confirmed by the use of an UV spectrophotometer. The UV spectrophotometer quantifies bacterial population through the turbidity of the broth culture. This procedure will indirectly indicate the absorbance of the light transmitted through the broth culture which stipulates if there has been any bacterial decay or growth in the sample. The focus of our team will be fixated on the change of acceleration in the death phase in the bacterial growth curve of E. coli due to our killswitch. We expect the death phase of our EC100 cells containing our Device 3 to be quicker than our control EC100 cells. As for the samples, we will generate three different controls: EC100 cells without any genetic circuit inoculated in a LB broth with Chloramphenicol, EC100 cells containing our Device 3 inoculated in a LB broth with Chloramphenicol, and EC100 cells without any genetic circuit inoculated in an nitrite and formaldehyde infused LB broth with Chloramphenicol. On the other hand, our experimental sample will be EC100 cells containing our Device 3 inoculated in an nitrite and formaldehyde infused LB broth with Chloramphenicol, since the inducers of the promoters present in the genetic circuit of Device 3 are nitrite and formaldehyde. After the data recollection we will perform a logarithmic growth curve of all of the samples.

Figure 3. UV Spectrophotometer Procedure.